Human Pituitary Prolactin Research Articles

Immunoglobulin G Subclasses and Prolactin (PRL) Isoforms in Macroprolactinemia Due to Anti-PRL Autoantibodies

Although macroprolactinemia due to antiprolactin (anti-PRL) autoantibodies is not uncommon among hyperprolactinemic patients, the pathogenesis of such macroprolactinemia is still unknown. We examined IgG subclasses of anti-PRL autoantibodies by enzyme immunoassay, and PRL phosphorylation and isoforms by Western blotting, mass spectrometry, and two-dimensional electrophoresis in six patients with anti-PRL autoantibodies and in 29 controls. PRL-specific IgG subclasses in patients with anti-PRL autoantibodies were heterogeneous, but five of six patients showed IgG4 predominance, which is known to be produced by chronic antigen stimulation. Western blot and mass spectrometric analyses revealed that human pituitary PRL was phosphorylated at serine 194 and serine 163, whereas serine 163 in serum PRL was dephosphorylated. On two-dimensional electrophoresis, serum PRL mainly consisted of isoform with isoelectric point (pI) 6.58 in control hyperprolactinemic patients, whereas acidic isoforms (pIs 6.43 and 6.29) were also observed in patients with anti-PRL autoantibodies. Our data first demonstrate that human pituitary PRL is serine phosphorylated and partially dephosphorylated in serum, and suggest that the acidic isoforms may give rise to chronic antigen stimulation in patients with anti-PRL autoantibodies.

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells.

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.

Prolactin as a local growth promoter in patients with locally advanced tongue cancer: GCRI experience

The purpose of this study was to assess the role of prolactin (PRL) in men with locally advanced tongue cancer. Circulating PRL was assayed immunoradiometrically in pretherapeutic and sequential blood samples of 99 patients with locally advanced tongue cancer. Patients were followed for 3 years or until their death within a stipulated time. Immunohistochemical localization of PRL was performed on formalin-fixed paraffin-embedded tissue sections. Tumoral prolactin receptors (PRLR) were estimated by ligand binding assay; the expression of PRL mRNA and PRLR mRNA were carried out by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, PRL amplimer was sequenced and compared with human pituitary PRL amplimer. Pretherapeutic PRL levels were significantly higher in patients with locally advanced tongue cancer compared with controls (p =.01). Thirty-four percent (34 of 99) of the patients had hyperprolactinemia (PRL >/=15.0 ng/mL). Univariate survival analysis showed that patients with pretherapeutic hyperprolactinemia had a significantly shorter overall survival than patients with pretherapeutic PRL <15.0 ng/mL serum (p =.0009). In multivariate analysis, PRL emerged as the most significant independent prognostic factor influencing overall survival. Furthermore, changes in serial PRL levels showed excellent correlation with response to therapy and progression of disease. Forty-four percent (24 of 54) of the tumors showed positive immunoreactivity with PRL antibody, indicating that PRL or a molecule similar to it is produced by tongue tumors. PRL mRNA expression was seen in 85% (43 of 50) of the tumors and confirmed the de novo synthesis of PRL. Sequence analysis of the 234 bp PRL amplimer revealed that the sequence was homologous to exon 5 of pituitary PRL mRNA. The action of PRL is mediated by PRLR, and it was observed that the PRLR positivity by ligand binding assay was 33%. The expression of PRLR mRNA by RT-PCR showed two forms of PRLR mRNA (ie, intermediate form [500-600 bp] seen in 82% (41 of 50 ) of the tumors and the long form [800-900 bp] seen in 36% (18 of 50) of the tumors. In 82% (41 of 50) of the tumors, either the intermediate or long form was seen. This multifaceted study of PRL suggests that tongue cancer cells produce PRL, and this ectopically produced PRL might be acting as a major local growth promoter by means of autocrine and paracrine mechanisms. Looking at its prognostic value and correlation with disease activity, it may provide new insights into treatment of tongue cancer.

Human prolactin gene expression. The use of an alternative noncoding exon in decidua and the IM-9-P3 lymphoblast cell line.

The prolactin (PRL) gene is normally expressed in the anterior pituitary lactotrope and the decidualized stromal cell of the human endometrium. We have recently described the ectopic expression of the human PRL gene in a B-lymphoblastoid cell line, IM-9-P. The IM-9-P and decidual PRL mRNAs are approximately 150 nucleotides longer than the pituitary transcript. The protein coding sequence of the IM-9-P and decidual PRL cDNAs is identical to the pituitary PRL message, and the 3'-untranslated region (UTR) exhibited a 7-14-nucleotide elongation. The 5'-UTR of IM-9-P and decidual PRL cDNAs revealed an extension of 41 base pairs upstream of the normal transcription start site for the human pituitary PRL gene. This is preceded by an additional sequence that is not homologous to the DNA found 5' to the pituitary PRL cap site. Decidual and IM-9-P PRL mRNAs possess identical 5' ends having 5'-UTRs of 83-263 nucleotides longer than that of the pituitary mRNA. The decidual/IM-9-P-specific segment of the 5'-UTR is located as a new 5'-noncoding exon 5-7 kilobase pairs upstream of the human pituitary PRL gene, exon 1. The clonal IM-9-P3 cell line provides a unique and easily manageable resource with which to study decidual specific PRL gene transcription.

The Human Placental Growth Hormone Variant Is Mitogenic for Rat Lymphoma Nb2 Cells*

Although there is evidence that human (h) placental GH variant (hGH-V) possesses a growth-promoting function, lactogenic activity by the hormone has not been demonstrated. Rat anterior pituitary tumor (GC) cells stably transfected with the hGH-V gene (GC [hGH-V] cells) synthesize and secrete hGH-V. This hormone shares considerable structural similarity with pituitary growth hormone (hGH-N) and chorionic somatomammotropin (hCS) at the nucleotide (greater than 90%) and amino acid (greater than 80%) levels. As expected, both hGH-N and hCS antibodies detect hGH-V by immunoblotting. However, hGH-V, but not hGH-N or hCS, cross-reacts with human or rat pituitary prolactin (PRL) antibodies. These data indicate that structural features shared by hGH-V and pituitary PRL are not present in hGH-N or hCS. Comparison of amino acid sequences implicates two regions that may account for a common epitope between hGH-V and hPRL, and structural difference from hGH-N and hCS. The possible lactogenic activity by hGH-V was assessed in a rat lymphoma Nb2 cell bioassay. Conditioned medium from GC[hGH-V] cells permitted growth of lactogen-dependent Nb2 lymphoma cells in culture. This activity was blocked by antibodies raised to rat PRL but not hPRL or hGH-N. Comparison of the hGH-V amino acid sequence with those from 14 other lactogenic hormones, including hPRL, hCS and hGH-N, reveals 6 conserved amino acids. These data indicate a lactogenic as well as growth-promoting function for the secreted hGH-V protein in vivo.

Observation of production of immunoactive prolactin by normal human connective tissue in cell culture

Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak or first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P less than or equal to 0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO approximately 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg.cell-1.3 d-1 feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. These in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia).

Fluid transport across human fetal membranes affected by human amniotic fluid prolactin: an in vitro study.

The aim of the study was to investigate the effects of human amniotic fluid (AF) prolactin (PRL) on the transfer of fluids across human fetal membranes, detected in vitro by weight change. We have developed a system, consisting of an inner compartment (4 ml), suspended in an outer compartment (40 ml), and separated from it by a 2 cm2 circular piece of fresh human fetal membrane (amnion, chorion and adhering decidua). The weight of the inner compartment was continuously registered with an electronic precision balance. Osmolality changes or hydrostatic pressure differences did not affect the rate of mass transfer through the membranes. Potassium cyanide significantly influenced the mass transfer in favour of the applied hydrostatic pressure difference (+2 cm H2O to the amniotic side) or osmotic pressure difference (amniotic side 265 mosm/kg/decidual side 285 mosm/kg). The PRL preparations used were human and ovine pituitary PRL, as well as three fractions isolated from human AF by fractionated ammonium sulphate and ethanol precipitations, followed by Sephacryl chromatography. All PRL preparations were tested in physiologic concentrations (0.5 to 5 micrograms/ml). Only the two largest AF-PRL variants significantly disturbed the balance of fluid transfer across the membranes when added to the fetal (amniotic) side of the membrane. This resulted in a net increase in fetomaternal transfer of 120 to 180 microliters. This effect could be repeated and lasted for at least 25 minutes. Using an antibody against hPRL the effect was completely blocked. Neither AF-PRL added to the maternal (decidual) side of the membrane nor oPRL or human pituitary PRL added to the amniotic or decidual side changed the rate of mass transfer across the membranes.

Physicochemical characterization and functional activity of fibroid prolactin produced in cell culture

Evidence from our laboratory with the use of cultured (primary and passaged) cells has extended our initial observation that human uterine fibroid is an extrapituitary source of prolactin. Fibroid prolactin antigen in conditioned medium reacted specifically in radioimmunoassay for human pituitary prolactin. Control experiments demonstrated that the radioimmunoassay results were not spurious due to degradation of tracer 125I-labeled prolactin. Immunoparallel dilution curves indicated antigenic relatedness of pituitary and fibroid prolactin. In a calibrated Sephadex G-100 column, fibroid prolactin eluted in the same region (20.3 to 20.9 kd) as purified pituitary prolactin. Glycosylated prolactin, detected by concanavalin A affinity column chromatography, appeared to constitute only a small percentage of fibroid prolactin made in culture. The ratio of fibroid prolactin bioactivity (lactogen Nb2 lymphoma bioassay) to antigen (radioimmunoassay) was 0.77. These data indicate that human uterine fibroid tissue produces a molecule similar to or, perhaps, identical with pituitary prolactin.

Stability studies on human pituitary prolactin.

Differently charged isomers of human pituitary prolactin were subjected to stability studies under various conditions. The study was guided by analytical electrophoresis and radioimmunoassay. In alkaline medium (pH 10) it was found that each isohormone was converted into faster-migrating components, probably due to deamidation. A quantitative study of this alteration was made by measuring the rate constant for this conversion assuming first-order kinetics. The result indicated that the rate constant decreased with increased acidity of the examined prolactin components. Furthermore, this alteration was also found to be paralleled by a decreased immunoactivity of the components. At neutral pH the conversion reaction was studied both in 0.9% NaCl and in human serum. For the least acidic isohormone the rate constant in serum was calculated to be about four times higher than that in the saline solution which in turn was comparatively low. It was concluded that the alteration observed at pH 10 might be attributed to non-enzymic deamidation. The conversion of prolactin that occurred in human serum indicated that this reaction could be caused by enzymes. A study of the immunoactivity of human prolactin as a function of time at various storage conditions is also included in this work. The stability of the hormone was found to be strictly concentration dependent and also affected by buffer ionic strength. In the presence of ethylene glycol (50%, v/v) at -20 degrees C and at a protein concentration in the range of 0.1-2 mg/ml the hormone was found to be both immunologically and electrophoretically stable for several years.

Cleaving of disulfide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis

Five highly purified preparations of human pituitary prolactin, widely used in radioimmunoassays, were analyzed by immunoperoxidase electrophoresis with rabbit antisera raised against ovine and rat prolactin. When unreduced, the prolactin content of these preparations was separated into one major prolactin-like immunoreactive band of M r 23,000, and four fainter immunoreactive bands of M r's 16,000, 22,000, 25,000, and 45,000. Increasing the 2-mercaptoethanol concentration modified the relative proportions and the M r of immunoreactive bands so that, after extensive reduction, only two immunoreactive bands were separated, one major band of M r 25,000 and one fainter band of M r 17,000. A direct relationship existed between the load of prolactin submitted to electrophoresis in the 0.25 to 75 ng range and the optical density of the separated bands.

Molecular cloning and nucleotide sequence of DNA complementary to human decidual prolactin mRNA.

Three human decidual prolactin (PRL) cDNA clones (pdPL-1:349 base pairs, pdPL-2:584 base pairs, pdPL-3:807 base pairs without poly(A) tract) were prepared and sequenced. The insert DNA of the largest clone pdPL-3 contained the coding region corresponding to 217 amino acid residues including 18 amino acid residues of the signal peptide, and 157 nucleotides of the 3'-untranslated region. A comparison of the pdPL-3 cDNA sequence with that of human pituitary PRL cDNA revealed 4 silent nucleotide differences. Two of the base changes occurred in the third position of the amino acid codons, and the other two occurred in the 3'-untranslated region. Therefore, the amino acid sequence of decidual PRL deduced from the nucleotide sequence of pdPL-3 was identical with that of pituitary PRL. Minor changes were also observed among the three PRL mRNAs: a silent change in the third codon in the translated region, and another change in the 3'-untranslated region. Southern blot hybridization of human cellular DNA with the pdPL-3 probe indicates that PRL gene may occur once per haploid genome. Therefore, these minor changes may reflect microheterogeneity of PRL gene. The existence of multiple poly(A)-adjacent sequences was shown in the three species of human decidual PRL mRNA and in human pituitary PRL mRNA. This variation may be due to heterogeneity in the processing at the 3' terminus of mRNA or the termination sites of the transcription.

The human fetal prolactin and estradiol levels, and their correlationship.

Serum prolactin and estradiol-17 beta levels were determined in umbilical cord sera obtained from 33 preterm, 118 full-term and 11 anencephalic infants and in peripheral sera obtained from 5 neonates. Prolactin levels increased from mid-gestation toward term, and markedly decreased after birth. Those in anencephalic infants (253 +/- 86 ng/ml, mean +/- S.D.) were lower than in full-term infants (324 +/- 119 ng/ml), but the difference was not statistically significant. We also performed thyroid-releasing-hormone (TRH) (50-200 micrograms) test on 5 anencephalic infants, but significant changes of serum prolactin levels were not found. Estradiol-17 beta levels in anencephalic infants were significantly lower than those in full-term infants. There was a significant positive correlation between cord prolactin and estradiol-17 beta levels in full-term infants. These results suggest one of the control factors for human fetal pituitary prolactin secretion is estrogen, and hypothalamic factors are not so important.

Synthesis of prolactin by human decidua in vitro.

Human decidua, chorion, amnion and placenta from the 1st., 2nd. and 3rd. trimesters of gestation were investigated for synthesis and secretion of prolactin by in vitro incubation of these tissue fragments in medium 199. Prolactin content in decidua was found to be significantly higher than that in chorion, amnion or placenta at any stages of gestation. During 6 hours of incubation, decidua secreted significantly more prolactin into medium than did chorion, amnion or placenta. The amount of prolactin secreted by decidua was significantly higher than the prolactin content in tissue before incubation. Decidua were also incubated in medium 199 with or without actinomycin-D (20-200 micrograms/ml), puromycin (200 micrograms/ml) or cycloheximide (100-200 micrograms/ml) for 12 hours. Both total prolactin secreted into medium and prolactin content in tissue after incubation were significantly lower than the control without inhibitor. Decidua of 2nd. trimester of gestation was noted to secrete more prolactin into medium than decidua of 1st. or 3rd. trimester of gestation. In further studies, 3H-leucine (200 muCi) was incubated for 12 hours with decidua (10 grams) from 2nd. trimester of gestation. The incorporated protein in medium or tissue was extracted, fractionated by gel filtration through Sephadex G-100 column and by 10% SDS-polyacrylamide gel electrophoresis. Peaks of 3H count and immunoreactive prolactin in gel slices were coincident with the peak of standard pituitary prolactin at gel slices' No. 31 (Rf = 0.62). These results demonstrate that prolactin is synthesized and secreted by human decidua, which is identical to human pituitary prolactin. The synthetic activity is most prominent in the decidua of the 2nd. trimester of pregnancy.

Purification and characterization of high molecular weight human pituitary prolactin.

Abstract A large form of human prolactin (molecular weight 150 000–170 000) was purified from the residue remaining after extraction at neutral pH of homogenized frozen pituitaries. This purification involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, and hydrophobic interaction chromatography on pentyl-Sepharose 4B. The procedure was followed by radioimmunoassay. The large form of prolactin was prepared both from fresh and from long-term stored residues. In the latter case the final yield was considerably higher. By zone electrophoresis in agarose suspension the prolactin preparation was separated into four or five immunoactive components. In sedimentation equilibrium analysis in the ultracentrifuge, however, these isohormones showed heterogeneity, which was suggested to be caused by dissociation. Evaluation of data obtained from the bottom region of the cells gave molecular weight values of the components in the range of 160 000 – 180 000. One of the is hormones s further s...

Human pituitary prolactin. Isolation and characterization of three isohormones with different bioassay and radioimmunoassay activities

Three forms of human pituitary prolactin, separable at alkaline pH in a highly purified preparation, were isolated by means of column electrophoresis in agarose suspension. The most acidic component showed a significantly lower radioimmunological activity but a higher bioactivity than the other two components, which were approximately equipotent in both assays. Consequently, in both assays the most acidic component differed markedly from the other components. Amino acid analysis indicated close similarity between the three components and no size heterogeneity was observed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel. A high degree of purity of the isolated components was demonstrated by analytical electrophoresis in polyacrylamide gel at alkaline pH. Runs in the same medium also revealed that the difference in electrophoretic migration velocity remained after reduction and alkylation of the isohormones. A comparison of the migrations of the S-carboxymethylated and the S-carbamidomethylated derivatives with those of the unmodified components indicated that the diversity in electrophoretical behaviour of two adjacent isohormones was consistent with a difference in one single net charge. By analytical isoelectric focusing the component of intermediate migration velocity was resolved into two distinct bands proposed to contain isohormones differing only in the exchange of one residue of aspartic acid for one of glutamic acid. The isoelectric points of the prolactin isomers were all in the range of 5.7–5.9.

Isolation of human pituitary prolactin

A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenizeed frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progresive purification was guided by radioimunoassays. The final product was obtained in yields of 31 μg/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination by growth hormone was low (less than 2%), and by other pituitary protein hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000–23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophores is the prolactin preparation was, however, heterogenous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.

Formula omitted][formula omitted] synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent in vitro protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When 3H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the 3H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue in vitro .

Culture of human pituitary prolactin and growth hormone cells.

Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9--13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1--2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.

Homologous radioimmunoassay for human prolactin

Radioimmunoassays for human prolactin in various biological fluids have been available since 1971. (1–7) Most of these assays have used heterologous systems with prolactin preparations from various sources, i.e. sheep, pig and primate with their corresponding antiserum. (1–5) Friesen et al. (6) find Sinha et al. (7) are the only authors who have described homologous assays using human prolactin as the reference standard and tracer as well and an anti-human prolactin antiserum. In the present study we have established a homologous radioimmunoassay using human pituitary prolactin. The separation method is based on the double antibody solid phase system. (8) Cross reactivity with human growth hormone (HGH), placental lactogen (HPL), the pituitary protein hormones and prolactins of various species were studied as were values found in normal subjects in basal conditions and after a TRH injection. The results of measuring basal levels and performing dynamic tests under various physiological and pathological circumstances will be reported subsequently.

Radioiodinated human pituitary and amniotic fluid prolactins with preserved molecular integrity;.

Human pituitary prolactin (hPRL) contains a single major component. Upon iodination by a lactoperoxidase procedure approximately 50% of this component remains ulaltered in molecular size and net charge by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Aminiotic fluid hPRL contains two components which remained ulaltered after iodination by the lactoperoxidase procedure used. Unaltered iodinated products can also be obtained from both sources by a stoichiometric chloramine T procedure, but in vastly diminished yield.